Production of penicillin



Patented Oct. 28, 1952 UNITED STATES tA ENT OFFICE 2.2515330 PRODUCTIoN oFr'ENicmLifi Commercial Solvents Corporation, Terre Haute, Ind., a corporation of Maryland;

Nol rawing. Application January 6, 1950,

selliitl N0. 137,269

The present invention relates to'a'pro'cess' for the preparation of penicillin. More particularly" it relates to a-process' for producing high yields'off Z CIaims-M (01. 195-36) penicillin bythe propagation of the-organism It is well nown thatthe organism P n i czll ium chrysogenum -.willsynthesize penicillin. when propagated in the presence of oxygen on various nutrient media, andthat theintroduction of certain factors into media will stimulate penicillin production, I 'lfhe fact-that oleic-acid and certain derivatives of thisa 'cid afiect the growth of oer-1,.

tairi bacteria, .bylstiniulatmn'under some'circuni- I stances and'by suppression of .gr'o wthfunder" others; is well known. For example, Du'bos and Davis. Jqniipu. Med. '83, 40943119467) observed that'son'i'e 'water-soluble esters tr m-1e, 'acid'exert a favorable e'fiect uponthefgrowth'ofthe tuberc e bacilli. The effect" of Y oleates upon" tlieigidwth and metabolic' ac'tiyity' otfun'g'iis'less known. At'

any? site," there seem's toflbe no means of p'r dieting thenutritional"requirements of a'nyorf-l dium, potassium, lithium, calciumand magnesi um oleates. Theincreases in penicillin-yields due to the presence of oleates in the media have been found us be most effective when'the oleic acid'or. metal ole'alte is" added" to the basal medium throughout the range o'fabout 0.002 to 0.4% based now discovered that oleic acid and oer Y toxicmetal salt's'oithat 'acid" promote or iiflibn" fof Penicillin y Penicillium chryso'gemlmin'th'e' presence of p 'jenyla'cetamide.

on the weight of the culturemedium. Best re- Any Qf 2- the various '6 procedures; employed conventional penicillinfermentations can :beiemplayed whenq using my. new medium; *T-his' in-:-

cludes the. conventional jaseptic techniquesg temperatures, rates of air flow through the media 2 and compositions of the media,'a's fermentation equipment. My metho I in increasing penicillin productior i'in Hi'EiiIa Qf widely varying types, both natural andsyzithetic', provided phenylacetamideis alse present? H My 'invention can be eitpla'inediin more detait by reference to the =following' specific examples which set": out the practical operating? 'embodi ments of my'process'bf-using o'l'eic acid oreol'eates'ii I and phenyla'cetamide'in the media."

MP L341" In .this -example'qa seriesof icompa rative was made to determine the effect uponpenicillin j production of varying concentrations; of sodium;-

ec ineyn me u sa lime i-l um;. used in each of ;these-tests-- contained thefollowing ingredienta inwater;

Percent by Weight Lactose 2.25 Glucose 0.75 NH4NO 0.50 Glacial acetic acid 0.25

KH2PO4 ..'.'0.20" 'Mgsoixmzoj i i 0.05 FeSOa'lI-IzO 0.02 ZnSO-4.7H2O e i -0.002

wee-4.51120 0.0005

The pH of-"the solution-was adjustedwith p0 tassium hydroxide to 5.9;" Onehundred'andfifty'" ml. of this basal medium -wasplaced -in- -each-of a series of one-liter Erlenmeyer flasks. Thebasal medium in some of the flasks was supplemented by the addition of "0.06% "by'weight' of penylacetamide and yarying-quantities of sodium oles :estishew in. t zta bel I h h m i was then sterilized and; inoculated with -an active' culture vof; Penicilli'u'm jysogenu'm'f and incu' bated for five days at 25- C2. on reciprocal shakers 1 toprovideaeration. -'l"'he results given in TjablefI below, show that an increase inpenicillinproduc tion o f, 11 3% over that obtained usi n'ga; bear: medium without phenylacet'ami de'or sodium me; ate, can be obtained when .06% phenylacetam'ide" and .04% sodium oleate are added. Also, an increase in yield of 50% is shown over that obtained with the basal medium plus .06% phenylacetamide-an increase of in yield is shown over the basal medium plus .06% sodium oleate alone.

Table I Additions to Basal Medium 3 days 4 days 5 days Na Oleate, Phenylacet- Penilfcni- Ifeni- Percent by amide, Percent pH cillin, pH 0111111, Cllhll, wt by wt. u./ml. u lml u./ml.

0.7 0 0 09 0.0 105 0.00 0.0 0 9 7.0 00 0. 00 0.7 50 0 9 00 7.0 150 0. 002 0.00 0.7 50 7 0 112 7.0 150 0. 00s 0.00. 7.0 02 7 1 7.1 177 0.01 0.00 7.0 70 7 0 102 0.9 100. 0.02 0.00 7.1 04 7 0 144 0.0 200 0. 04 0. 00 7.0 so 7 0 140 0.9 224 0.00 0.00 0.0 r 91 7 0 140 0.0 100 0.1 0. 00 7.0 100 7 0 144 0.0 100 0.15 0.00 0.0 100 0 0 153 0.0 104 EXAMPLE II Table III A series of media was prepared, each medium 1 a Assay Gms Crude consisting of: v Fermentation ho. ulmL Penicillin 360 gallons corn steep liquor 023 7, 415 1500 pounds lactose 070 5,420 80 pounds phenylacetamide 25 gallons corn oil 000 41202 11 gallons oleic acid 232 r 11 gallons mineral oil. 55 D I Water to 10,000 gallons I 290 5, 5-59 The pH of each medium was adjusted to 5.'75.9 Ave 370 v 5,960 with soda ash. Each medium was then sterilized,

' Table II Assay gins. Crude I v Fermentation l\0. ulmL Penicillin.

EXAMPLE III 1 A seriesof fermentationswas run exactlyaccording to the procedure described in Example II,

except that the media employed did not contain any oleic acid. The results of this series of fermentations are shown in Table III. As is evident from a comparison of the results shown in Tables II and III, the addition of oleic acid to fermentation media brings about considerable in creases in penicillin yields. v

While I have described what I consider to be the most advantageous modifications of my moo-- ess, it is evident of course that various modifications can be made in the specific procedures which have been described without departing from the purview of this invention.

Iclaim: 1. Ina process for preparing'penicilli n by'th propagation of the organism Pe'mc'tllium chrysog genum in a nutrient medium. the improvementv which comprises propagating said organism under aerobic conditions in a nutrient medium containing from about 0.002 to 0.8% by weight'of pl ien ylacetamide and about 0.002 to 0.4% by weight of a member selected from the group consisting'of oleic acid and non-toxic metal oleates. V

under aerobic conditions in a nutrient medium containing from about 0.002 to 0.8%v by weight of phenylacetamide and from about 0.002 to 0.4%. 1 by weight of sodium oleate. p j I I RICHARD, J HICKEY.

REFERENCES CITED The following references are of record in the i 1 le of this patent:

UNITED STATES PATENTS 1 OTHER REFERENC S Penicillin Research Progress ReportNo. May 7 27, 1944, Departments of Biochemistry etc; University of Wisconsin, W. P. B. Contract '118, page 2.

Ibidum, Report No. 10; August 5', 1944, page 3. I

Stefaniak et al.: Journal of Bacteriology, vol.- 52, No. 1, July 1946, pages 119, to 127. 

1. IN A PROCESS FOR PREPARING PENICILLIN BY THE PROPAGATION OF THE ORGANISM PENICILLIUM CHRYSOGENUM IN A NUTIENT MEDIUM, THE IMPROVEMENT WHICH COMPRISES PROPAGATING SAID ORGANISM UNDER AEROBIC CONDITIONS IN A NEUTRIENT MEDIUM CONTAINING FROM ABOUT 0.002 TO 0.8% BY WEIGHT OF PHENYLACETAMIDE AND ABOUT 0.002 TO 0.4% BY WEIGHT OF A MEMBER SELECTED FROM THE GROUP CONSISTING OF OLEIC ACID AND NON-TOXIC METAL OLEATES. 